The ability to treat neuropathic pain (NP) remains unsatisfactory, as many patients experience pain despite taking prescribed medications. Although opioids are effective against NP, their clinical usefulness is limited by their central nervous system effects (i.e., sedation, cognitive dysfunction, and tolerance). Using the L5 spinal nerve ligation model in rodents, we found that loperamide, a peripherally acting opioid, alleviates behavioral signs of NP. In addition, we reported that enhancing <-opioid receptor (MOR) expression in primary afferents, using HSV viral vectors, results in a leftward shift in the analgesic dose-response curve of loperamide. Thus, opioid receptors in the peripheral nervous system present a valuable target for the treatment of NP in humans. Surprisingly, repetitive administration of loperamide induces peripheral opioid tolerance. In this proposal, we aim to modulate the interactions of MORs and -opioid receptors (DORs) in the peripheral nervous system to enhance peripheral opioid analgesia and to attenuate peripheral opioid tolerance. We hypothesize that after nerve injury, the functional interactions between MORs and DORs are crucial for both the observed peripheral opioid antihyperalgesic effects and the development of peripheral opioid tolerance. Specifically, we hypothesize that: 1) DOR agonists enhance the analgesic effects of peripheral MOR agonists in opioid-naove animals with NP, 2) chronic administration of a MOR agonist switches DORs from an enhancing to an inhibitory role, thereby contributing to peripheral opioid tolerance, and 3) the development of tolerance is due to the formation of MOR-DOR heterodimers, and peripheral opioid tolerance in NP can be prevented or reversed by inhibiting heterodimerization. A broad range of strategies will be used to test these hypotheses, including behavioral, electrophysiological, Ca2+ imaging, and molecular biological methods. We will test whether concurrent activation of peripheral MORs and DORs will result in enhanced antihyperalgesia in opioid-naove rodents with NP (Aim 1) and whether repeated administration of peripheral MOR agonist switches the DOR into a negative modulator of MOR function, resulting in attenuation of peripheral opioid antihyperalgesia (Aim 2). To study the role of DOR and DOR-MOR heterodimerization in opioid antihyperalgesia and peripheral opioid tolerance, innovative virus-mediated gene transfer strategies will be used to suppress DOR expression and to inhibit DOR-MOR heterodimerization by expressing a dimer-deficient DOR (Aim 3). In addition, a novel small peptide that blocks DOR-MOR heterodimerization will be studied for its effect on opioid tolerance. The results of these studies will provide new insights into the mechanisms that underlie the peripheral pain-reducing effects of opioids in NP and the mechanisms that underlie peripheral opioid tolerance. Furthermore, the use of virus mediated gene transfer and small cell permeable peptide inhibitors of heterodimerization are viable pre-clinical approaches to develop novel therapeutic strategies for the treatment of NP and opioid tolerance.